Prevention of infection by highly pathogenic viruses using topical application of povidone-iodine on mucous membranes

ABSTRACT

This invention is directed to methods for prevention of infections by highly pathogenic viruses by applying to the nasal mucous membranes topical preparations comprising the broad-spectrum antimicrobial agent povidone-iodine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Patent Cooperation Treatyapplication serial number PCT/AU2020/050586, filed Jun. 10, 2020, whichclaims priority to Australian patent application no. 2019902006, filedJun. 10, 2019, and Australian patent application no. 2020900489, filedFeb. 20, 2020, the contents of each of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

This invention is directed to methods for prevention of infections byhighly pathogenic viruses by applying to the nasal mucous membranestopical preparations comprising the broad-spectrum antimicrobial agentpovidone-iodine.

BACKGROUND OF THE INVENTION

Diseases caused by, generally zoonotic, highly pathogenic viruses are ofincreasing concern to health authorities. Prominent recent examples arethe outbreaks of diseases caused by the SARS and MERS coronaviruses(SARS-CoV, MERS-CoV), the avian influenza virus (AIV), Ebola virus(EBOV) and the virus responsible for COVID19 (SARS-CoV-2). Withincreasing globalization, the threat of diseases caused by such highlypathogenic viruses has been amplified. In some cases, there is the addedconcern that the viruses could be used for bioterrorism.

For the purposes of this invention, the term highly pathogenic virusesor HP viruses will be used, where the term includes all viruses wherethere is limited access to effective treatment or vaccines, becausethere are currently no effective treatment or vaccines or where theyexist but deployment of such interventions is compromised orcontraindicated (for example, the use of treatments that may beunsuitable for patients with other concomitant morbidities or there maybe delays in vaccine deployment) and where the viruses are capable ofcausing serious disease, including fatal disease, in humans if untreatedand/or significant societal impact, including the potential to be usedas bioterror weapons.

By way of specific example, HP viruses will include the following:Filoviridae able to cause fatal viral haemorrhagic fever (VHF) inhumans, notably EBOV, Marburg virus (MARV), Sudan virus (SUDV), TaiForest virus (TAFV) and Bundibugyo virus (BDBV); coronaviruses able tocause serious or fatal illness in humans notably MERS-CoV, SARS-CoV andSARS-CoV-2; and avian influenza virus (AIV) and other emergent strainsof influenza with pandemic potential. It will be understood that otheremergent viruses of similar potential impact and concern may beidentified in the future, either as a result of a marked change in thetransmission potential of a known virus or emergence of a new species orstrain.

Apart from the fact that all these viruses are capable of causingserious and fatal illness in humans and that an effective treatment orvaccine may not yet exist, or be contraindicated and/or difficult torapidly deploy, the other important commonality is that they are allzoonotic, i.e. originally transmitted from animals to humans. By way ofexample, the animal source for AIV is birds, and for EBOV it is believedto be fruit bats.

Another common feature is that they have relatively high mortality ratesin humans and in many cases are readily transmissible, often via therespiratory route. Typically these viruses will be categorised as level3 or 4 risk groups and in many cases defined as biological select agentsby regulatory authorities such as in the USA. In some cases, such asnovel, emergent influenza viruses and SARS-CoV-2, they have thepotential to become pandemic, causing significant societal impact, largenumbers of deaths and major challenges for frontline healthcare workerswho are at risk of contracting the virus. A further concern is that someof the viruses responsible for these diseases may have the capacity tobe developed as bioterror weapons.

Ebola

Ebola viruses are of particular concern. They cause a VHF that is knownas Ebola virus disease (EVD) or simply Ebola, which in 2014 killed morethan 9,000 people in West Africa and caused global concern. For thepurposes of this invention, the term “Ebolavirus” will be deemed toinclude all five filoviruses viruses capable of causing human VHF,namely EBOV, MARV, SUDV, TAFV and BDBV.

Fruit bats are believed to be the natural reservoir of EBOV and contactwith bat faeces or other body fluids may be responsible for the initialinfection of humans, after which human-human transmission can occur. Asnoted by Osterholm et al (“Transmission of Ebola Viruses: What We Knowand What We Do Not Know.” mBio, 6, 2 (2015): 1-9) human-to-humantransmission of EBOV occurs principally via direct contact with blood orbodily fluids from an infected person who is showing signs of infectionor by contact with objects recently contaminated by an actively illinfected person.

Bosio et al (“Ebola and Marburg Viruses Replicate in Monocyte-DerivedDendritic Cells without Inducing the Production of Cytokines and FullMaturation.” The Journal of Infectious Diseases 2003; 188:1630-8)reported that the profound virulence of EBOV and MARV is due to theirability to infect and replicate in dendritic cells and monocytesundetected until the numbers of virus are beyond the ability of the hostimmune response to contain the infection. The infected immune cells thenpropagate the infection through the body.

Mohomadzadeh et al (“How Ebola and Marburg viruses battle the immunesystem.” Nature Reviews Immunology 7, (2007): 556-567) also reportedthat productive infection, that is infection that results in more viralprogeny being produced, occurs primarily in dendritic cells, monocytesand macrophages.

Osterholm et al (2015) hypothesized that Ebolaviruses have the potentialto be respiratory pathogens with primary respiratory spread byaerosolization of droplet micronuclei following coughing and vomiting byEbola sufferers. EBOV has been found on the outside of face masks wornby health workers caring for victims of the disease and it has beenestablished that the virus can infect certain cells of the respiratorytract.

Prescott et al (“Postmortem Stability of Ebola Virus.” EmergingInfectious Diseases, Vol. 21, No. 5, May 2015: 856-859), reported thatbased on animal models the virus can be isolated from corpses for atleast seven days after death, with large numbers of virus found in thenasal and oral cavities.

Osterholm et al (2015) also reported that the infectious dose forEbolaviruses in humans appears to be extremely low, with ten or fewerviral particles being sufficient for infection placing healthcareworkers at risk despite barrier prevention procedures.

Because of these factors, and other factors, such as environmentalconditions and training, standard barrier protection procedures, such ashand hygiene and personal protective equipment (PPE) such as gloves,masks, eye protection and respirators, may not be sufficient to preventinfection of healthcare workers from Ebola. This is evidenced by theWorld Health Organization's review of healthcare worker infectionsduring the 2013-16 Ebola epidemic and publication of the draftdiscussion paper “Preferred Product Characteristics for PersonalProtective Equipment for the Healthcare Worker on the FrontlineResponding to Ebola Virus and Haemorrhagic Fever Outbreaks in TropicalClimate (WHO 7 Sep. 2017). Meanwhile, those exposed without PPE, such asfamily members, are at high risk of infection. Therefore, the needexists to better protect healthcare works and civilians who might beexposed to Ebolaviruses, when exposed to infected people and their bodyfluids.

Pandemic Influenza

Like Ebola, pandemic influenza is a potentially fatal illness thatcontinues to cause global concern. It is caused by of the introductionof an influenza virus with high pathogenic potential into a naive humanpopulation that has no pre-existing immunity and often whose naturalhost is an animal—for example, birds in the case of avian influenzavirus (AIV) and pigs in the case of swine influenza—or a recombinantvirus containing human virus derived genes together with genes notpreviously found in human isolates. One of the most highly pathogenicstrain of AIV to date is denoted H5N1 and has been spreading throughoutAsia since 2003. Other known and potentially highly pathogenic strainsare H5N9, H7N3, H7N7, H7N9, and H9N2. The most recent 2009 pandemic“swine flu” virus (generally believed to be a reassortment of bird,swine, and human flu viruses) is designated H1N1, specifically(H1N1)pdm09 and is reported to have been associated with 151,700-575,400deaths worldwide in the first year of the pandemic. It is generallybelieved that most initial human infections of AIV are a result ofeither handling dead infected birds or from direct contact with infectedfluids. However, once a person is infected, the risk of human-humantransmission is of notable concern possibly by analogy with other humaninfluenza viruses through aerosolization of the virus during coughingand sneezing and subsequent inhalation by others. Human-humantransmission can also occur through contact of mucous membranes (forexample of the eyes or nasal passages) with body fluids and touchingobjects contaminated with the virus.

Outbreaks of AIV and other emergent influenza viruses are managed bypractices such as the elimination of the infected animals, isolation ofinfected people, screening at airports for people with influenzasymptoms, wearing of facemasks by civilians who perceive they might beat risk, and adherence to strict barrier protection and infectioncontrol procedures by healthcare workers, including hand hygiene andpersonal protective equipment (PPE) such as gowns, gloves, eyeprotection and respirators.

Existing antiviral agents such as zanamivir and oseltamivir have been oflimited effectiveness in treating AIV infections, because by the timethe patient presents it is generally too late in the infection. Further,the virus has demonstrated the ability to mutate and thereby circumventsuch drugs, particularly oseltamivir, in some cases. Efforts are alsounderway to develop vaccines but no effective vaccine for all pandemicstrains has become available to date and, despite many attempts, nouniversal vaccine has been developed.

Given these considerations and the fact that the virus is spread throughairborne transmission, there is the grave concern that AIV and relatedinfluenza viruses could be used as a bioterror weapon.

The need exists to better protect healthcare workers and civilians whomight be exposed to emergent influenza viruses of high pathogenicity,either when handling infected animals or when exposed to infectedpeople.

SARS and MERS

Like AIV, Severe Acute Respiratory Syndrome or SARS is a potentiallyfatal illness caused primarily by airborne transmission of a virus andprimary infection of the respiratory tract. SARS is caused by acoronavirus, called SARS-associated coronavirus or SARS-CoV.Coronaviruses are a family of single-stranded enveloped RNA viruses thatare divided into four major genera. The genome sequence of SARS-CoV-2 is82% similar to severe acute respiratory syndrome coronavirus (SARS-CoV),and both belong to the β-genus of the coronavirus family. Humancoronaviruses such as SARS-CoV and Middle East respiratory syndromecoronavirus (MERS-CoV), are known to cause respiratory and entericsymptoms. SARS was first reported in Asia in February 2003. The illnessspread to more than two dozen countries before the global outbreak of2003 was finally contained. During 2003, a total of 8,098 peopleworldwide were infected with SARS and 774 died. In late 2019 the worldwitnessed the emergence of the novel coronavirus (SAR-CoV-2) whichcaused a new acute respiratory syndrome designated COVID-19 and whichwas later designated as a global pandemic.

Early symptoms include a high fever, headache, malaise and body aches.Some also have mild respiratory symptoms or diarrhoea. After 2 to 7days, SARS patients may develop a dry cough. Many patients developpneumonia, which is the principal cause of mortality.

SARS-CoV is transmitted by respiratory droplets produced when aninfected person coughs or sneezes, with the infected droplets depositedon the mucous membranes of the mouth, nose, or eyes of people who arenearby. The virus also can spread when a person touches a surface orobject contaminated with infectious droplets and then touches his or hermouth, nose, or eyes.

MERS is also a serious and often fatal respiratory illness in humanscaused by a novel coronavirus, designated MERS-CoV. MERS was first foundin the Arabian Peninsula and believed to originate in camels. BetweenSeptember 2012 and Jun. 5 2015, there had been 1279 human cases of MERSreported and 495 deaths, or a mortality rate of 38.7%. The disease wasfirst reported in Saudi Arabia but more recently was found in Korea andmore than 20 other countries.

MERS-CoV, like other coronaviruses, is thought to spread from aninfected person's respiratory secretions, such as through coughing.Early symptoms may include fever, chills, coughing, shortness of breath,body aches, sore throat, headache, diarrhoea, nausea and/or vomiting,and runny nose. However, a wide clinical spectrum of MERS-CoV infectionhas been reported, ranging from asymptomatic infection to acute upperrespiratory illness, and rapidly progressive pneumonitis, respiratoryfailure, septic shock and multi-organ failure resulting in death.

Those at primary risk of infection include frontline healthcare workersand the families of those infected. Those at secondary risk includeairline flight crews, Emergency Medical Service (EMS) units at airports,customs and immigration personnel and international travellers. Forhealthcare workers, recommended protection includes hand hygiene,gloves, gowns, eye protection and respirators.

For both SARS and MERS, recommended protection for frontline healthcareworkers includes hand hygiene and personal protective equipment (PPE)such as gowns, gloves, eye protection and respirators. However, suchprotections may not be fully effective and may not be available to allhealthcare workers and others who may be exposed.

Protection from Highly Pathogenic (HP) Viruses

Unbroken epidermal skin provides an effective barrier against theinvasion of microbes into the human body, including invasion by HPviruses. However, mucous membranes, such as in the eyes, nasal passages,oral cavity and lungs are more permissive and so provide a morereceptive surface for microbial ingress and systemic infection. Thenasal passages also provide an effective entrapment mechanism to preventparticles and aerosols, including microbes, from being transportedfurther along the airways to the bronchioles and lungs. This filtrationmechanism arguably results in accumulation of viruses and bacteria inthe nasal passages where they are able to proliferate, requiring thebody to mount an immune defence to counter the burgeoning viral load.The escalating viral load within the nasal passages increases the riskof the virus finding its way into the lungs, such as in MERS and SARS,or spreading systemically through the body.

SUMMARY OF THE INVENTION

The present invention involves methods for reducing the risk oftransmission or preventing infection by HP viruses by application ofPVP-I preparations to the nasal passages of subjects who have beenexposed to or may be exposed to HP viruses, this would also includereducing the risk of infection or transmission of infection from peoplesuspected of shedding HP viruses (for example, people believed to beinfected or at risk of being infected as a consequence of prior exposureto a potential source of infection).

In all cases, the methods involve the application of pharmaceuticalpreparations to the nasal passages of human subjects, said preparationscomprising at least 0.10% w/v and no more than 1.25% w/v PVP-I. In allcases, the application may occur before, after or around the time ofexposure of the subject to a person or people infected (or maybeinfected) with HP viruses or other sources of infection. In all cases,the applications occur at a frequency of between 1 and 12 times dailyand continue at a suitable daily frequency for a number of days afterany such exposure depending on the incubation period for infectioncaused by each virus.

Accordingly in one aspect, the present invention provides a method forreducing the risk of infection or transmission of infection by EBOVcapable of causing Ebola virus disease, by application of a PVP-Ipreparation comprising between 0.10% w/v and 1.25% w/v PVP-I to thenasal passages of a subject exposed or potentially exposed to EBOV, saidapplication occurring with a frequency of between 1 and 12 times dailycommencing before, after or around the time of exposure to the virusand/or thereafter for preferably up to 21 days.

In another aspect the present invention provides a method for reducingthe risk of infection or transmission of infection by filoviruses otherthan EBOV, including MARV, SUDV, TAFV and BDBV, capable of causing viralhaemorrhagic fevers, by application of a PVP-I preparation comprisingbetween 0.10% w/v and 1.25% w/v PVP-I to the nasal passages of a subjectexposed or potentially exposed to the filoviruses, said applicationoccurring with a frequency of between 1 and 12 times daily commencingbefore, after or around the time of exposure to the virus and/orthereafter for preferably up to 21 days.

In another aspect the present invention provides a method for reducingthe risk of infection or transmission of infection by the avianinfluenza viruses (AIV) designated H5N1 that is capable of causingpandemic influenza, by application of a PVP-I preparation comprisingbetween 0.10% w/v and 1.25% w/v PVP-I to the nasal passages of a subjectexposed or potentially exposed to H5N1, said application occurring witha frequency of between 1 and 12 times daily commencing before, after oraround the time of exposure to the virus and/or thereafter forpreferably up to 8 days.

In another aspect the present invention provides a method for reducingthe risk of infection or transmission of infection by pandemic influenzaviruses other than H5N1 including H5N9, H7N3, H7N7, H7N9, H9N2 and H1N1that are capable of causing serious or pandemic influenza, byapplication of a PVP-I preparation comprising between 0.10% w/v and1.25% w/v PVP-I to the nasal passages of a subject exposed orpotentially exposed to the viruses, said application occurring with afrequency of between 1 and 12 times daily commencing before, after oraround the time of exposure to the virus and/or thereafter for up topreferably 21 days, such as up to 8 days.

In another aspect the present invention provides a method for reducingthe risk of infection or transmission of infection by SARS-CoV capableof causing Severe Acute Respiratory Syndrome (SARS), by application of aPVP-I preparation comprising between 0.10% w/v and 1.25% w/v PVP-I tothe nasal passages of a subject exposed or potentially exposed toSARS-CoV, said application occurring with a frequency of between 1 and12 times daily commencing before, after or around the time of exposureto the virus and/or thereafter for up to preferably 21 days, such as upto 10 days.

Accordingly in one aspect, the present invention provides a method forreducing the risk of infection or transmission of infection bySARS-CoV-2 capable of causing COVID-19 disease by application of a PVP-Ipreparation comprising between 0.10% w/v and 1.25% w/v PVP-I to thenasal passages of a subject exposed or potentially exposed toSARS-CoV-2, said application occurring with a frequency of between 1 and12 times daily commencing before, after or around the time of exposureto the virus and/or thereafter for up to preferably 21 days.

In another aspect the present invention provides a method for reducingthe risk of infection or transmission of infection by MERS-CoV capableof causing Middle East Respiratory Syndrome (MERS), by application of aPVP-I preparation comprising between 0.10% w/v and 1.25% w/v PVP-I tothe nasal passages of a subject exposed or potentially exposed toMERS-CoV, said application occurring with a frequency of between 1 and12 times daily commencing before, after or around the time of exposureto the virus and/or thereafter for up to preferably 21 days.

In another aspect the invention provides a method of decreasing therelease of a HP virus from the respiratory tract, primarily from thenasal passages, of a person infected with the HP virus, the methodcomprising application to the nasal passages of the infected person aneffective amount of a pharmaceutical preparation comprisingpovidone-iodine (PVP-I) at a concentration of greater than 0.10% w/v andless than about 1.25% w/v. For instance, the present inventors haveshown marketed reduction of the replication of SARS-CoV-2 in thepresence of the PVP-I formulation according to the present invention. Inthis regard the reduction of measurable viral growth after PVP-Iexposure was observed to be at least −2.4 Log10 which represents areduction in viral RNA copies of over 99%.

It is evident from the Figures and experimental contained herein thatSARS-CoV-2 replication was effectively eliminated in the samples exposedto PVP-I, as evidenced by the lack of detection of virus-specific RNA insamples grown on Vero cells compared to untreated control samples. Thesedata correspond to a reduction of viable, replication competentSARS-CoV-2 by >99.97% compared to control values. Notably, andsurprisingly, the virus associated RNA in the inoculum samples was notdepleted by PVP-I treatment. These data highlight that a) PVP-I did notinterfere with the RT-PCR assay method and therefore the RNA detectiondata are valid; b) it is essential that a method involving culture ofthe virus, as described in the present example, be used to detect theeffect of PVP-I on virus viability. In the absence of a culture step toestablish viral replication competence the effect of PVP-I may have beenobscured.

It is taught herein that the application may occur with a frequency ofbetween 1 and 12 times daily commencing before, after or around the timeof exposure to the virus and/or thereafter for up to 21 days (or for theperiod of suspected shedding should symptoms persist and/or there is anyother concern, for example a positive diagnostic test, that the subjectmay still be shedding).

In another aspect the invention provides a method of decreasing theimpact of secondary infections of a person infected with a primary HPvirus, the method comprising application to the nasal passages of theinfected person an effective amount of a pharmaceutical preparationcomprising povidone-iodine (PVP-I) at a concentration of greater than0.10% w/v and less than about 1.25% w/v. Said application occurring witha frequency of between 1 and 12 times daily commencing after or aroundthe time of exposure to the primary virus and/or thereafter for up topreferably 14 days (or for the period symptoms associated with theprimary and/or secondary persist and/or there is any other concern, forexample a positive diagnostic test, that the subject may still beinfected).

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Detection of SARS-CoV-2 RNA in 96-well plate supernatants byreal-time TaqMan RT-PCR post exposure to test solutions. SARS-CoV-2 wasexposed to the indicated test solution(s) for 1 minute before serialdilution (1:3) and incubation on Vero cells for 48 h. Values expressedas mean cycle threshold (Ct) value+SEM (n=3) versus dilution factor (1:3increments) (A) Time point zero (0 h) inoculum titration used todetermine baseline Ct-values of treated samples. (B) Culturesupernatants harvested 48 h post-inoculation on to Vero cells.

FIG. 2: Detection of SARS-CoV-2 RNA in 96-well plate supernatants byreal-time TaqMan RT-PCR post exposure to test solutions. SARS-CoV-2 wasexposed to the indicated test solution(s) for 1 minute before serialdilution (1:3) and incubation on Vero cells for 96 h. Values expressedas mean cycle threshold (Ct) value+SEM (n=3) versus dilution factor (1:3increments) (A) Time point zero (0 h) inoculum titration used todetermine baseline Ct-values of treated samples. (B) Culturesupernatants harvested 96 h post-inoculation on to Vero cells.

DETAILED DESCRIPTION OF THE INVENTION

To date, PVP-I has never been proposed as a preventative for infectionscaused by HP viruses by application to the nasal passages. Its use as atopical antiseptic on hands and skin to prevent infection by viruses,including HP viruses, is known and widely applied as part of barrierregimens for preventing access of the virus to the interior of the humanbody.

Australian patent application 2014206143 to Molloy and Goodall,discloses that application of PVP-I to the nasal passages can be used totreat and prevent infections caused by common cold viruses, but there isno suggestion that a similar application to the nasal passages couldprevent infection and/or shedding by/of HP viruses or diseasesassociated with secondary infections, nor is it obvious because SARS,MERS and AIV, for example, are considered lower respiratory tractinfections (LRI) with very different clinical presentation and pathologyto an infection limited to the upper respiratory tract (URI) such as thecommon cold. Furthermore Ebola is considered to be a consequence ofsystemic infection.

The present invention discloses for the first time that application ofPVP-I to the nasal passages has utility in the reduction of risk ofinfection and transmission of infection, the prevention of illness aswell as the decrease risk of secondary infections caused by andassociated with HP viruses such as Ebolaviruses, such as EBOV,pathogenic coronaviruses, such as SARS-CoV and MERS-CoV, and pandemicinfluenza viruses, such as AIV, and other emergent HP viruses.

EBOV

In the case of EBOV and other filoviruses, whether the exposure occursby direct contact with infected body fluids or through airborne virus,at some point the virus needs to gain access to the internal human bodyin order to initiate and propagate the infection. A likely portal forsuch access is the mucous membranes of the face, the respiratory tractand in particular the nasal passages.

As is the case with the common cold, the eyes may be one portal, ascould occur when someone rubs their eyes with fingers contaminated withvirus. However due to the physiology of the human body it is most likelythat any virus entering through the eyes would find its way to the nasalpassages and replicate there as part of the process of establishing aproductive infection.

The oral cavity might also be a useful portal for the virus but theconstant secretion of saliva and excretory mechanisms such as swallowingwould argue against this as an important access point for the virus. Incontrast, the nasal passages represent a highly receptive area for aninfection and in the absence of a cold or other nasal infection,secretions are limited and as taught by Australian patent application2014206143 to Molloy et al, previously referenced herein, normalmucociliary clearance takes around 15 minutes. The nasal passages offeranother important advantage for filoviruses such as EBOV in that thenasal passages contain monocytes that act as sentinel cells forinfection, also as taught in Australian Patent 2014206143 to Molloy etal, previously referenced herein. Such immune cells are known to becomeinfected by EBOV and to act as a ‘Trojan horse’ for the EBOV infectionthat spreads to organs throughout the body. These immune cells are notcommonly found in the oral cavity.

Further the presence of EBOV or EBOV RNA in the nasal passages wouldcause the monocytes to release cytokines thereby attracting neutrophilsto the nasal passages, which would also be susceptible to infection byEBOV and in fact, rather than only fight the infection, which is theirnormal role, it is postulated that their infection would accelerate andpropagate the infection along with the infected monocytes.

Therefore, the present invention discloses for the first time thatthrough the unexpected combination of these factors, the nasal passagessurprisingly offer a uniquely attractive portal for an initial entry ofEBOV into the human host as a prelude to systemic replication anddisease. However, it is this point of entry that also makes EBOVespecially susceptible to attack and destruction by PVP-I throughapplication to the nasal passages.

It is known that PVP-I rapidly destroys EBOV and likely all otherfiloviruses at low PVP-I concentrations. Also, as taught in AustralianPatent 2014206143 to Molloy et al previously referenced herein, it isknown that PVP-I is toxic to immune cells such as monocytes at low PVP-Iconcentrations. This knowledge combined with the disclosure above of themechanism of ingress and infection by EBOV has led the current inventorsto a new discovery and that is that the repeated intranasal applicationof PVP-I according to the inventive method would not only eliminate anyEBOV present in the nasal passages before an infection can be initiatedbut would destroy any infected monocytes or neutrophils and eliminatenon-infected monocytes or neutrophils present so that could not becomesubsequently infected by EBOV. In essence, PVP-I application to thenasal passages would transform the nasal passages from a convenientportal of access for EBOV to a secondary barrier of protection from EBOVinfection, greatly reducing the overall risk of EBOV infection.

Australian Patent 2014206143 to Molloy et al previously referencedherein teaches that for use in the nasal passages, a suitableconcentration of PVP-I is between 0.10% w/v and 2.5% w/v, with the lowervalue representing the level below which there is little or no effectiveantimicrobial capacity and the upper value representing the level abovewhich PVP-I is known to cause ciliotoxicity in the nose. For use in thetreatment of the common cold the patent teaches against the use ofliposomal preparations of PVP-I, as defined in the patent, because oftheir slower performance and reduced antiviral activity in the face ofmucin inactivation, secretory clearance and a most resilient virus,human rhinovirus. In the case of Ebolaviruses, however, which areenveloped viruses and far more sensitive to PVP-I, and with the absenceof the rhinorrhoea present in colds, these factors are less applicable.Indeed the slower release and longer residence time associated withliposomal PVP-I formulations, or other slow release/longer residencetime forms, for example gels may be an advantage for preventativeapplications such as EBOV infection. Therefore, the current inventiondoes not exclude liposomal formulations of PVP-I.

As to frequency and timing of application, this will depend on thecircumstances of the exposure to EBOV or similar viruses. In the case ofhealthcare workers who are using PPE and associated barrier techniqueswhile they are exposed to infected individuals, it is not practical toapply a preparation to the nose while the PPE are in place and to do somay increase exposure to viruses. The first opportunity to apply a PVP-Iintranasal preparation could be before or while applying PPE (i.e., aspart of a PPE “suiting up” regime) or could be at the time of removingthe PPE after completing work activities, where the purpose of theapplication of intranasal PVP-I is to eliminate any viruses that mightsomehow have circumvented the PPE and other barriers, for example due tofailure of a respirator or by the worker inadvertently touching theexterior of a face mask or gown during removal and subsequently touchingtheir nose or eyes allowing the virus to potentially find its way to thenasal passages. In those circumstances, the worker should use theproduct immediately after removing the PPE in accordance withrecommended safe work practices and thereafter at a frequency of up to12 times daily for the period equivalent to the incubation period forthe virus, which in the case of EBOV is up to 21 days after the mostrecent exposure to the virus.

In the case of people who do not have PPE and may be exposed to virus,such as family members, the PVP-I intranasal preparation would need tobe used continuously during the exposure period at a frequency of up to12 times daily and after the exposure at a similar frequency for theperiod equivalent to the incubation period for the virus, which in thecase of EBOV is up to 21 days after the most recent exposure to thevirus.

In every case, the volume of the PVP-I intranasal preparation should besufficient to reach all parts of the nasal passages, which in the caseof a liquid PVP-I intranasal preparation as taught in Australian Patent2014206143 to Molloy et al previously referenced herein may represent avolume of up to 1 mL applied to each nostril of the exposed person.

The PVP-I intranasal preparation may be in the form a solution, drops,spray, gel, cream, aerosol, or inhalant.

AIV

In the case of AIV and as disclosed by Shinya et al (“Influenza virusreceptors in the human airway.” Nature, 440: 435-6) H5N1 onlyproductively replicates in the cells of the lower respiratory tract, notthe nasal passages. Therefore, the application of PVP-I to the nasalpassages would not be regarded by one skilled in the art as a productivemeans of treating or preventing AIV infection.

However, the present inventors have discovered that where human-humantransmission occurs, it would normally occur as the result of thesneezing or coughing of an infected person, with the virus carried bydroplets. Such droplets are large enough to deposit in the nasalpassages and while the virus may not replicate in the nasal cells, thenasal passages are expected to act as an important initial staging pointfor establishing infection and/or prior to further ingress to the lowerrespiratory tract. For example, the virus could be carried bymucociliary clearance to the throat from where it can readily migrate tothe bronchi.

Further, in the case of animal-human transmission of AIV, as might occurwith people tending, handling or otherwise exposed to animals,especially poultry, infected with AIV, the virus would be carried ondust and other particles and also very likely find its way to the nasalpassages and lodge there prior to further migration into the lungs.

In either animal-human or human-human transmission, the virus could alsobe carried on hands and the person may self-inoculate by touching theireyes or nose with contaminated fingers. Again, the nasal passages couldbe important in ferrying the virus ultimately to the lungs.

Therefore, the present invention discloses for the first time thatwhether a person is exposed to infected animals or humans, theapplication of PVP-I to the nasal passages provides an importantprotective effect against infection and/or replication/shedding of virusimmediately post infection.

It is known that PVP-I rapidly destroys H5N1 and all other influenzaviruses, pandemic or otherwise, at low PVP-I concentrations. Thisknowledge combined with the disclosure above of the mechanism of ingressand infection by AIV has led the current inventors to a new discoveryand that is that the repeated intranasal application of PVP-I accordingto the inventive method would eliminate any AIV present in the nasalpassages before the virus can migrate to the lower respiratory tract andestablish a productive infection. In essence, the nasal application ofPVP-I would transform the nasal passages from a convenient portal ofaccess for AIV to a secondary barrier of protection from AIV infection,greatly reducing the overall risk of AIV infection.

For reasons already discussed, a suitable concentration of PVP-I isbetween 0.10% w/v and 1.25% w/v, such as about 0.15%, 0.2%, 0.25%, 0.3%,0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%,0.9%, 0.95%, 1%, 1.05%, 1.1%, 1.15%, or about 1.2% or any range withinsuch concentrations.

As to frequency and timing of application, similar considerations applyto those already discussed for EBOV. In the case of animal or healthcareworkers who are using PPE and associated barrier techniques while theyare exposed to AIV infected animals or people, the worker should use theproduct before or while applying PPE or immediately after removing thePPE and thereafter at a frequency of up to 12 times daily for the periodequivalent to the incubation period for the virus, which in the case ofAIV may be as long as 17 days.

In the case of people who do not have PPE and may be exposed to virus,such as family members, the PVP-I intranasal preparation would need tobe used continuously during the exposure period at a frequency of up to12 times daily and after the exposure at a similar frequency for theperiod equivalent to the incubation period for the virus, which in thecase of AIV is up to 17 days after the most recent exposure to thevirus.

In every case, the volume of the PVP-I intranasal preparation should besufficient to reach all parts of the nasal passages, which in the caseof a liquid PVP-I intranasal preparation may represent a volume of up to1 mL applied to each nostril of the exposed person.

The PVP-I intranasal preparation may be in the form a solution, drops,spray, gel, cream, aerosol, or inhalant.

HP Coronaviruses (e.g., SARS and MERS)

The MERS-CoV causes a LRI, which can result in severe pneumonia withacute respiratory distress and multiple-organ failure leading to a highmortality rate. Like AIV, transmission is likely through airbornedroplets or direct or indirect contact with the virus.

Adney et al (“Replication and Shedding of MERS-CoV in Upper RespiratoryTract of Inoculated Dromedary Camels.” Emerging Infectious Diseases, 20,12 (2014): 1999-2005) showed that MERS-CoV actively replicated in theupper respiratory tract of camels.

Memish et al (“Middle East respiratory syndrome coronavirus infectionsin health care workers.” N Engl J Med 369 (2013):884-886) reportedasymptomatic carriers of MERS CoV often had mild URI symptoms as wellsuggesting that the virus might infect the nasal passages of humans, butin a subsequent study Memish et al (“Prevalence of MERS-CoV NasalCarriage and Compliance With the Saudi Health Recommendations AmongPilgrims Attending the 2013 Hajj.” JID 210 (2014): 1067-1072) found noevidence of nasal carriage of MERS-CoV, at least in asymptomaticsubjects.

For these reasons and the fact that SARS and MERS are considered lowerrespiratory tract infections, the application of PVP-I to the nasalpassages would not be regarded by one skilled in the art as a productivemeans of treating or preventing SARS or MERS infections.

However, where human-human transmission occurs, it would normally occuras the result of the sneezing or coughing of an infected person, withthe virus carried by droplets. Such droplets are large enough to depositin the nasal passages and while the virus may not replicate in the nasalcells, the nasal passages may act as an important initial staging pointfor further ingress to the lower respiratory tract. For example, thevirus could be carried by mucociliary clearance to the throat from whereit can readily migrate to the bronchi.

Further, in the case of animal-human transmission of SARS or MERS, asmight occur with people tending, handling or otherwise exposed toinfected animals, the virus would be carried on dust and other particlesand also likely find its way to the nasal passages and lodge there priorto further migration into the lungs.

In either animal-human or human-human transmission, the virus could alsobe carried on hands and the person may self-inoculate by touching theireyes or nose with contaminated fingers. Again, the nasal passages couldbe important in ferrying the virus ultimately to the lungs. Furthermore,Sungnak and colleagues have recently reported evidence that cells of thenasal passages may be particularly susceptible to infection and havepostulated a potential role in initial viral infection, spread andclearance of SARS-CoV-2 (Sungnak, et al., 2020. Nature Comms. 26:681-687. “SARS-CoV-2 entry factors are highly expressed in nasalepithelial cells together with innate immune genes”).

Therefore, the present invention discloses for the first time thatwhether a person is exposed to infected animals or humans, theapplication of PVP-I to the nasal passages may provide an importantprotective effect against ingress to the lower respiratory tract andproductive infection. This would be especially important for health careworkers and other susceptible people who could be exposed to infectedindividuals which have tested positive, for instance, for SARS-CoV-2,display the symptoms of COVID-19 disease, or for people who are testingindividuals to determine if they are positive for the virus

The examples provided herein show that PVP-I also inactivates SARS-CoV-2(FIGS. 1 and 2).

This knowledge combined with the disclosure above of the mechanism ofingress and infection by SARS or MERS coronaviruses has led the currentinventors to a new discovery and that is that the repeated intranasalapplication of PVP-I according to the inventive method would eliminateany SARS-CoV or MERS-CoV present in the nasal passages before the viruscan migrate to the lower respiratory tract and establish a productiveinfection.

This would include “suppressing’ viral infection in an individualinfected, for instance with SAR-CoV-2 or other viruses, in order toreduce the risk of transmission.

The concept of “suppressing” viral infection indicates any aspect ofviral infection, such as viral replication, time course of infection,amount (titer) of virus, lesions, and/or one or more symptoms iscurtailed, inhibited, or reduced (in terms of severity and/or duration)in an individual or population of individuals treated with a PVP-Icomposition in accordance with the invention as compared to an aspect ofviral infection in an individual or population of individuals nottreated in accordance with the invention. Reduction in viral titerincludes, but is not limited to, elimination of the virus from aninfected site or individual. Viral infection can be assessed by anymeans known in the art, including, but not limited to, measurement ofvirus particles, viral nucleic acid or viral antigens, detection ofsymptoms and detection and/or measurement of anti-virus antibodies.Anti-virus antibodies are widely used to detect and monitor viralinfection and generally are commercially available. In addition, viralinfection can be assessed by other means known in the art including, butnot limited to, PCR, in situ hybridization with virus specific probes,TCID₅₀ assays, infectious center assays, plaque assays, etc.

For reasons already discussed, a suitable concentration of PVP-I isbetween 0.10% w/v and 1.25% w/v, such as about 0.15%, 0.2%, 0.25%, 0.3%,0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%,0.9%, 0.95%, 1%, 1.05%, 1.1%, 1.15%, or about 1.2% or any range withinsuch concentrations.

As to frequency and timing of application, similar considerations applyto those already discussed for EBOV and AIV. In the case of animal orhealthcare workers who are using PPE and associated barrier techniqueswhile they are exposed to AIV infected animals or people, the workershould use the product prior to or at the same time as applying PPE, orimmediately after removing the PPE and thereafter at a frequency of upto 12 times daily for the period equivalent to the incubation period forthe virus, which in the case of SARS is up to 10 days and for MERS is upto 14 days.

In the case of people who do not have PPE and may be exposed to virus,such as family members, the PVP-I intranasal preparation would need tobe used continuously during the exposure period at a frequency of up to12 times daily and after the exposure at a similar frequency for theperiod equivalent to the incubation period for the virus, or up to 10days for SARS and 14 days for MERS after the most recent exposure to thevirus.

In every case, the volume of the PVP-I intranasal preparation should besufficient to reach all parts of the nasal passages, which in the caseof a liquid PVP-I intranasal preparation may represent a volume of up to1 mL applied to each nostril of the exposed person.

The PVP-I intranasal preparation may be in the form a solution, drops,spray, gel, cream, aerosol, or inhalant.

In relation to the HP viruses discussed above in certain embodiments thenasal application provides a clinically significant Log reduction of atleast several log units (e.g. 2-4 log units) within about 5-20 minswhich persists for about 8 hrs after being exposed to said virus afterone application within the nasal passage. In an embodiment thecomposition provides a clinically significant Log reduction of at leastseveral log units (e.g. 2-4 log units) within about 5-20 mins whichpersists for about 6 hrs after being exposed to said virus after oneapplication within the nasal passage. In an embodiment the compositionprovides a clinically significant Log reduction of at least several logunits (e.g. 2-4 log units) within about 5-20 mins which persists forabout 4 hrs after being exposed to said virus after one applicationwithin the nasal passage. In another embodiment the composition providesa clinically significant Log reduction of at least several log units(e.g. 2-4 log units) within about 5-20 mins which persists for 2 hrsafter being exposed to said virus after one application within the nasalpassage. In another embodiment the composition provides a clinicallysignificant Log reduction of at least several log units (e.g. 2-4 logunits) within about 5-20 mins which persists for 1 hr after beingexposed to said virus after one application within the nasal passage.

In relation to the HP viruses discussed above in certain embodiments thenasal application provides a clinically significant Log reduction of atleast several log units (e.g. 2-4 log units) within about 10 mins whichpersists for about 8 hrs after being exposed to said virus after oneapplication within the nasal passage. In an embodiment the compositionprovides a clinically significant Log reduction of at least several logunits (e.g. 2-4 log units) within about 10 mins which persists for about6 hrs after being exposed to said virus after one application within thenasal passage. In an embodiment the composition provides a clinicallysignificant Log reduction of at least several log units (e.g. 2-4 logunits) within about 10 mins which persists for about 4 hrs after beingexposed to said virus after one application within the nasal passage. Inanother embodiment the composition provides a clinically significant Logreduction of at least several log units (e.g. 2-4 log units) withinabout 10 mins which persists for 2 hrs after being exposed to said virusafter one application within the nasal passage. In another embodimentthe composition provides a clinically significant Log reduction of atleast several log units (e.g. 2-4 log units) within about 10 mins whichpersists for 1 hr after being exposed to said virus after oneapplication within the nasal passage.

Secondary Infections

It is well known that so called “secondary infections” can be a commoncomplication of a primary viral infection, particularly in the case of aprimary respiratory viral disease. These secondary infections are oftenbacterial infections. Secondary infections occurs during or after aninfection from another pathogen, commonly viruses. These secondarydiseases are thought to be facilitated by a number of factors, includingviral mediated enhanced attachment and colonization, viral mediatedenhanced pathogenesis and viral mediated immune-modulation. Secondaryinfections are responsible for increased morbidity and in some casesmortality and have been shown to increase the societal and individualimpact of HP viruses. For example, during the H1N1 2009 influenzapandemic secondary infection by a variety of micro-organisms includingMycoplasma pneumoniae, S. aureus, K. pneumoniae, S. pneumoniae, M.catarrhalis, P. aeruginosa, S. pyogenes, and Streptococcus agalactiaewas shown to be present in approximately a third to a half of allfatalities (Morris et al., Front. Microbiol. 2017; 8: 1041 “SecondaryBacterial Infections Associated with Influenza Pandemics” and referencestherein). Similarly secondary infections have been commonly reportedfollowing autopsy of fatal COVID-19 disease there are concerns thatthese may contribute to mortality and morbidity associated with therecent pandemic (Cox et al., Lancet, Microbe 2020; 1: E11“Co-infections: potentially lethal and unexplored in COVID-19”, andreferences therein). These secondary infections present a complexdiagnostic challenge since they may comprise a variety of species andmay prove difficult to resolve from micro-organisms colonizing thepatient benignly or in some cases previously benign organisms may becomepathogenic following the primary infection. PVP-I is reported to havebroad spectrum anti-microbial activity and rapidly inactivates a widerange of micro-organisms including antibiotic resistant strains(Kanagalingam, et al., Int J Clin Practice. 2015 69; 11: 1247-1256,“Practical use of povidone-iodine antiseptic in the maintenance of oralhealth and in the prevention and treatment of common oropharyngealinfections” and references therein). The use of PVP-I has been proposedas a universal “disinfectant” for the purposes maintaining, for example,oral health associated with microbial infections. Importantly, due toits mode of action and broad spectrum activity it is not expected thatknowledge of the identity or antibiotic susceptibility of themicro-organism responsible for the secondary infection will be requirednor the lack of this knowledge limit the utility of PVP-I.

Secondary infections, often associated with bacteria and fungi, are aknown cause of serious morbidity and mortality in viral diseases,particularly respiratory viruses. Secondary infections are also thoughtto have played a role in recent pandemics caused by HP viruses. They areoften difficult to diagnose and, for example in the case of antibioticresistant organisms, can pose treatment challenges. The primary virusinfection is thought to predispose a patient to secondary diseasesthrough, for example, promoting colonization, immune-modulation andother factors such as facilitating increased virulence. Diseases such asbacterial pneumonia caused by secondary infections are a result ofbacterial infection of the lower respiratory tract.

For these reasons the application of PVP-I to the nasal passages wouldnot be regarded by one skilled in the art as a productive means oftreating or preventing secondary infections.

Therefore, the present invention discloses for the first time thatwhether a person is exposed to infection with a HP virus, theapplication of PVP-I to the nasal passages may provide an importantprotective effect against secondary infections and the diseasesassociated with secondary infections. This would be especially importantfor health care workers and other susceptible people who may be atincreased risk.

It is known that PVP-I has a broad spectrum antimicrobial effect (2006),(previously referenced herein), and is unaffected by antibioticresistance. In some cases, the organisms involved in secondary diseasesmay colonise the nasal passages in a benign fashion prior to the primaryinfection.

This knowledge combined with the disclosure above of the mechanism ofingress and infection by HP viruses has led the current inventors to anew discovery and that is that the repeated intranasal application ofPVP-I according to the inventive method would eliminate micro-organismspresent in the nasal passages, thereby preventing the promotion ofcolonization and exacerbated disease, and limit their spread before theycan migrate to the lower respiratory tract (or elsewhere) and establisha productive secondary infection.

For reasons already discussed, a suitable concentration of PVP-I isbetween 0.10% w/v and 1.25% w/v, such as about 0.15%, 0.2%, 0.25%, 0.3%,0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%,0.9%, 0.95%, 1%, 1.05%, 1.1%, 1.15%, or about 1.2% or any range withinsuch concentrations.

As to frequency and timing of application, similar considerations applyto those already discussed for primary infection with HP viruses. In thecase of animal or healthcare workers who are using PPE and associatedbarrier techniques while they are exposed to HP virus infected animalsor people, the worker should use the product prior to or at the sametime as applying PPE, or immediately after removing the PPE andthereafter at a frequency of up to 12 times daily for the periodequivalent to the incubation period for the virus.

In the case of people who do not have PPE and may be exposed to virus,such as family members, the PVP-I intranasal preparation would need tobe used continuously during the exposure period at a frequency of up to12 times daily and after the exposure at a similar frequency for theperiod equivalent to the incubation period for the virus.

In every case, the volume of the PVP-I intranasal preparation should besufficient to reach all parts of the nasal passages, which in the caseof a liquid PVP-I intranasal preparation may represent a volume of up to1 mL applied to each nostril of the exposed person.

It is intended that the preparations be applied at “ambient temperature”which refers to the temperature in the environment at which the methodof the current invention is conducted. Typically ambient temperaturewill be about 10° C. to about 30° C. Importantly the term “ambienttemperature” means that neither the formulation nor the nasal passagesof the subject to be treated are exposed to external heating in carryingout the method of the present invention.

Intranasal Preparations

As disclosed herein the PVP-I intranasal preparations may be in the forma solution, drops, spray, gel, cream, aerosol, or inhalant.

Suitable concentration of PVP-I is between 0.10% w/v and 1.25% w/v, suchas about 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%,0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.1%,1.15%, or about 1.2% or any range within such concentrations.

In certain embodiments the PVP-I intranasal preparations are aqueousbased intranasal preparations where water makes up from about 80% w/v toabout 96% w/v of the total preparation.

In certain embodiments the PVP-I preparations disclosed hereinadditionally includes an amount of iodide and/or iodate salts of fromabout 0.003 to about 0.05% w/v of the total preparation.

In certain embodiments the PVP-I preparations disclosed herein may alsoinclude one or more of the following: solubilising agents, polarsolvents, dry acidulant, sequestrants, alkaline agents, counter-irritantagents, local anaesthetic and preservatives.

In certain embodiments the PVP-I intranasal preparations are aqueousbased intranasal preparations where water makes up from about 80% w/v toabout 96% w/v of the total preparation and has a pH of from about 2-7such as a pH range of about 3-6.

In certain embodiments the PVP-I preparations disclosed hereinadditionally includes an amount of humectant such as hyaluronic acid,polyethylene glycol or glycerol (glycerine) from about 1 to about 10%w/v of the total preparation.

In certain embodiments the PVP-I preparations disclosed hereinadditionally includes an amount of a polar solvent, such as ethanol,from about 0.2 to about 2% w/v of the total preparation.

In certain embodiments the PVP-I preparations disclosed hereinadditionally includes an amount of a counter-irritant or localanaesthetic, such as menthol, from about 0.001 to about 1% w/v of thetotal preparation.

In certain embodiments the PVP-I preparations disclosed hereinadditionally includes an amount of a preservative, such as a quaternaryammonium salt preservative, from about 0.01 to about 0.5% w/v of thetotal preparation.

In certain embodiments the PVP-I preparations disclosed hereinadditionally includes an amount of buffer such as sodium phosphate,citrate or acetate from about 0.05 to about 0.5% w/v of the totalpreparation.

The pharmaceutical preparation may further comprise at least onepharmaceutically acceptable diluent, excipient or carrier.

The diluent, excipient or carrier may be a flavor agent, sweetener,colouring agent, solvent, buffer, alcohol, polymer, surfactant or otherdiluent, excipient or carrier designed to optimize the nasal delivery,intranasal distribution, stability, effectiveness, acceptability,tolerability of the preparation.

EXAMPLES Aqueous-Intranasal Preparation Examples Example 1

Per 100 mL g % w/v PVP-I 0.50  0.50% Potassium Iodide 0.010 0.010%Hyaluronic Acid 1.00  1.00% Sodium Citrate 0.15  0.15% Eucalyptus 0.010.010% Ethanol 0.49 0.490% Benzalkonium Chloride 0.01  0.01% Water 97.8397.83% 100.00 100.0%

Example 2

Per 100 mL g % w/v PVP-I 0.50  0.50% Potassium Iodide 0.010 0.010%Potassium Iodate 0.005 0.005% Glycerol 5.00  5.00% Sodium HydrogenPhosphate 0.15  0.15% Ethanol 0.49  0.49% Menthol 0.01 0.010% Water93.84 93.84% 100.00 100.0%

Example 3

Per 100 mL g % w/v PVP-I 0.50  0.50% Potassium Iodide 0.010 0.010%Potassium Iodate 0.005 0.005% Glycerol 5.00  5.00% Sodium HydrogenPhosphate 0.15  0.15% Sodium Hydroxide 0.75  0.75% Ethanol 0.49  0.49%Menthol 0.01 0.010% Water 93.08 93.08% 100.00 100.0%

Example 4

Per 100 mL g % w/v PVP-I 0.50  0.50% Povidone 0.300  0.30% Glycerol 3.00 3.00% Sodium Citrate 0.15  0.15% Citral 0.49  0.49% Water 95.56 95.56%100.00 100.0%

Example 5

Per 100 mL g % w/v PVP-I 0.50  0.50% Povidone 0.030 0.030% PotassiumIodide 0.010 0.010% Polyethylene Glycol 4000 2.00  2.00% Tween 20 0.05 0.05% Sodium Hydrogen Phosphate 0.15  0.15% Ethanol 0.49  0.49%Peppermint Oil 0.01 0.010% Water 96.76 96.76% 100.00 100.0%

Example 6

Per 100 mL g % w/v PVP-I 0.50  0.50% Sodium Iodide 0.010 0.010% SodiumAcetate 0.15  0.15% Sorbitol 0.01 0.010% Lutrol 0.50 0.500% Water 98.8398.83% 100.00 100.0%

Example 7

Per 100 mL g % w/v PVP-I 0.50  0.50% Lutrol 2.00  2.00% Sodium Citrate0.15  0.15% Saccharine 0.01 0.010% Water 97.34 97.34% 100.00 100.0%

Example 8

Per 100 mL g % w/v PVP-I 0.50  0.50% Polyethylene Glycol 4000 3.00 3.00% Sodium Hydrogen Phosphate 0.15  0.15% Xylitol 1.00  1.00% Water95.35 95.35% 100.00 100.0%

Example 9

Per 100 mL g % w/v PVP-I 0.50  0.50% Triacetin 2.00  2.00% SodiumAcetate 0.15  0.15% Tween 20 0.05  0.05% Ethanol 0.49  0.49% EucalyptusOil 0.01 0.010% Water 96.80 96.80% 100.00 100.0%

Example 10

Per 100 mL g % w/v PVP-I 1.00   1.00% Potassium Iodide 0.02   0.02%Hyaluronic Acid 0.50   0.50% Sodium Citrate 0.15   0.15% Eucalyptus 0.01  0.01% Ethanol 0.49   0.49% Benzalkonium Chloride 0.01   0.01% Water97.82  97.82% 100.00 100.00%

Example 11

Per 100 mL g % w/v PVP-I 1.00 1.00% Potassium Iodide 0.02 0.02%Potassium Iodate 0.01 0.01% Glycerol 5.00 5.00% Sodium HydrogenPhosphate 0.15 0.15% Ethanol 0.49 0.49% Menthol 0.01 0.01% Water 93.3293.32% 100.00 100.00%

Example 12

Per 100 mL g % w/v PVP-I 1.00 1.00% Povidone 0.60 0.60% Glycerol 3.003.00% Sodium Citrate 0.15 0.15% Citral 0.49 0.49% Water 94.76 94.76%100.00 100.00%

Example 13

Per 100 mL g % w/v PVP-I 1.00 1.00% Povidone 0.06 0.06% Potassium Iodide0.02 0.02% Polyethylene Glycol 4000 2.00 2.00% Tween 20 0.05 0.05%Sodium Hydrogen Phosphate 0.15 0.15% Ethanol 0.49 0.49% Peppermint Oil0.01 0.01% Water 96.22 96.22% 100.00 100.00%

Example 14

Per 100 mL g % w/v PVP-I 1.00 1.00% Sodium Iodide 0.02 0.02% SodiumAcetate 0.15 0.15% Sorbitol 0.01 0.01% Lutrol 0.50 0.50% Water 98.3298.32% 100.00 100.00%

Example 15

Per 100 mL g % w/v PVP-I 1.00 0.50% Lutrol 2.00 2.00% Sodium Citrate0.15 0.15% Saccharine 0.01 0.01% Water 96.84 96.84% 100.00 100.00%

Example 16

Per 100 mL g % w/v PVP-I 1.00 0.50% Polyethylene Glycol 4000 3.00 3.00%Sodium Hydrogen Phosphate 0.15 0.15% Xylitol 1.00 1.00% Water 94.8594.85% 100.00 100.00%

Example 17

Per 100 mL g % w/v PVP-I 1.00 0.50% Triacetin 2.00 2.00% Sodium Acetate0.15 0.15% Tween 20 0.05 0.05% Ethanol 0.49 0.49% Eucalyptus Oil 0.010.01% Water 96.30 96.30% 100.00 100.00%

Example 18

Per 100 mL g % w/v PVP-I 0.20 0.20% Potassium Iodide 0.02 0.02%Hyaluronic Acid 0.50 0.50% Sodium Citrate 0.15 0.15% Eucalyptus 0.010.01% Ethanol 0.49 0.49% Benzalkonium Chloride 0.01 0.01% Water 98.6298.62% 100.00 100.00%

Example 19

Per 100 mL g % w/v PVP-I 0.20 0.20% Potassium Iodide 0.02 0.02%Potassium Iodate 0.01 0.01% Glycerol 5.00 5.00% Sodium HydrogenPhosphate 0.15 0.15% Ethanol 0.49 0.49% Menthol 0.01 0.01% Water 93.3293.32% 100.00 100.00%

Example 20

Per 100 mL g % w/v PVP-I 0.20 0.20% Povidone 0.60 0.60% Glycerol 3.003.00% Sodium Citrate 0.15 0.15% Citral 0.49 0.49% Water 95.56 95.56%100.00 100.00%

Example 21

Per 100 mL g % w/v PVP-I 0.20 1.00% Povidone 0.06 0.06% Potassium Iodide0.02 0.02% Polyethylene Glycol 4000 2.00 2.00% Tween 20 0.05 0.05%Sodium Hydrogen Phosphate 0.15 0.15% Ethanol 0.49 0.49% Peppermint Oil0.01 0.01% Water 97.02 97.02% 100.00 100.0%

Example 22

Per 100 mL g % w/v PVP-I 0.20 1.00% Sodium Iodide 0.02 0.02% SodiumAcetate 0.15 0.15% Sorbitol 0.01 0.01% Tween 20 Lutrol 0.50 0.50% Water99.12 99.12% 100.00 100.0%

Example 23

Per 100 mL g % w/v PVP-I 0.20 0.50% Lutrol 2.00 2.00% Sodium Citrate0.15 0.15% Saccharine 0.01 0.01% Water 97.64 97.64% 100.00 100.00%

Example 24

Per 100 mL g % w/v PVP-I 0.20 0.50% Polyethylene Glycol 4000 3.00 3.00%Sodium Hydrogen Phosphate 0.15 0.15% Xylitol 1.00 1.00% Water 95.6595.65% 100.00 100.00%

Example 25

Per 100 mL g % w/v PVP-I 0.20 0.50% Triacetin 2.00 2.00% Sodium Acetate0.15 0.15% Tween 20 0.05 0.05% Ethanol 0.49 0.49% Eucalyptus Oil 0.010.01% Water 96.30 96.30% 100.00 100.00%

Biological Data a) SARS-CoV-2

PVP-I nasal intranasal preparation of the examples inactivatesSARS-CoV-2 in a cell culture system

Introduction

This study assessed the ability of PVP-I to inactivate SARS-CoV-2, shownto replicate efficiently within the upper respiratory tract. PVP-I wasassessed against saline and media only controls in parallel to assessthe presence/absence of virucidal activity.

Study Objective

The objective of this study is to compare and assess the virucidalactivity of PVP-I against the SARS-CoV-2 strain(BetaCoV/Australia/VIC01/2020) in the African Green Monkey Kidney (Vero)human cell line.

The impact on virus replication will be qualitatively assessed forSARS-CoV-2 RNA using a SARS specific TaqMan Real-time PCR assaytargeting the E-gene.

Materials Virus

SARS-CoV-2 (BetaCoV/Australia/VIC01/2020) was isolated and grown by theVictorian Infectious Diseases Reference Laboratory from a positivepatient specimen in January, 2020. Whole-genome sequencing confirmed thepresence of SARS-CoV-2 (GenBank ID: MT007544).

Placebo and Povidone Iodine Solutions

PVP-I examples according to the description were prepared at aconcentration of PVP-I between 0.1% w/v and 1.25% w/v (designated as“PVP-I”—this examples section)

Cells

Vero cells were grown in Eagle's Minimum Essential Medium(Sigma-Aldrich, North Ryde, Australia) catalogue number (M2279),supplemented with 1× Non-essential amino acids (NEAA) (Gibco, MountWaverley, Australia; catalogue number 11140050) and 10% heat-inactivatedfoetal bovine serum (FBS: Bovogen, Melbourne, Australia; cataloguenumber SFBS, lot# 1502B).

Methods Plating 96-Well Plates

Vero cells were trypsinised, counted and seeded into 96-well plates at acell density of 1×104 cells/well in 200 ul media and incubated at 37°C., 5% CO₂. Outer perimeter wells of the plate were not used, tominimise potential edge effects.

Incubation of Virus with Treatment Solutions

95 ul of each test solution (saline or PVP-I) was incubated with 5 ul ofeach virus at 37° C. for 1 minute. At the end of incubation, the testsolution/virus mix was diluted 1:10 with ice cold EMEM 2% FBS media.Immediately after adding ice cold media, 100 ul of test mixture for thesamples was added to the plates in triplicate (column 2 below). Note:PVP-I was tested in duplicate (PVP-I #1, PVP-I #2). Samples werethoroughly mixed and 100 ul transferred to column 3 with fresh pipettetips. 100 ul was transferred from column 3 to column 4 and so on untilcolumn 10. 100 ul from column 10 was discarded, leaving a final volumeof 200 ul in the wells. Column 11 contained cells alone with fresh EMEM2% which served as a no virus control. Plates were incubated at 37° C.,5% CO2 atmosphere for 48 hours before harvesting.

Uninfected dilution factor: 3 9 27 81 243 729 2187 6561 19683 log(10)dilution 0.5 1 1.4 1.9 2.4 2.9 3.3 3.8 4.3 CONTROL 2 3 4 5 6 7 8 9 10 ASample 1 B C D Sample 2 E F G H

Purification of SARS-CoV RNA

SARS-CoV-2 RNA was purified from 200 ul supernatant/media from each wellof the 96-well plates using the QIAamp 96 virus QlAcube HT Kit andprocessed on the QIAcube robotic extraction platform (QIAgen, Hilden,Germany) to confirm the presence/absence of replicating virus.

Taqman RT-PCR of SARS-CoV RNA

Purified SARS-CoV-2 RNA was reverse transcribed to cDNA using theBioline Sensifast cDNA kit (Catalogue number CSA-01148; London, UK).Real-time assays were performed using the published SARS E-gene assay[1]with primers and probes (IDT, Singapore) and ABI TaqMan Fast UniversalPCR Master Mix (2×) (catalogue number 4352042; Thermofisher, Vilnius,Lithuania). Assays were performed on a Thermofisher ABI 7500 Fast RealTime PCR machine.

Results Taqman RT-PCR Assessment of SARS-CoV-2 RNA in 96-Well PlateSupernatants

To confirm that the presence or absence of replicating SARS-CoV-2, 200ul supernatant samples from all triplicate wells were analysed for thepresence of SARS-CoV-2 RNA by Taqman RT-PCR.

To establish a baseline of RT-PCR cycle threshold (Ct) values fornon-replicating, non-viable virus, initial inoculum samples (0 h postinfection) were titrated 1:3 (˜0.5 log10) to 1:19683 (4.3 log10) andsubjected to the E-gene specific Taqman RT-PCR. SARS-CoV-2 alone, in thepresence of saline, and Nasodine, show a linear relationship betweenCt-value and dilution factor (see FIGS. 1A, 2A). Overlapping values wereobserved for all conditions, indicating that the test solutions did nothave any effect in the ability of our assay to detect nucleic acid byreal-time RT-PCR.

48 Hr Study

Following 48 h of incubation on Vero cells, SARS-CoV-2 displayed robustreplication both within and without the presence of saline (FIG. 1B).When incubated in the presence of PVP-I, an almost linear relationshipwas observed between viral RNA Ct-values and dilution factor (whereCt-values increased as the dilution factor increased), at similar levelsto inoculum only controls, indicating the absence of any measurableviral growth after PVP-I treatment and a reduction in detectable by atleast −2.4 Log10 in the PVP-I samples, representing a reduction in viralRNA copies of over 99%.

Conclusion of 48 H Study

The objective of this study was to compare and assess the virucidalactivity of PVP-I solution against the newly emerged SARS-CoV-2, thecausative agent of COVID-19 disease.

Using real-time TaqMan RT-PCR methods it was determined that PVP-Ipossesses clear virucidal activity against SARS-CoV-2 (−2.4 Log10reduction, or >99% kill) compared to saline alone.

96 Hr Study

Following 96 h of incubation on Vero cells, SARS-CoV-2 in media alonedisplayed robust replication (FIG. 2B), as shown by a decrease inCt-value. When incubated in the presence of Nasodine, an almost linearrelationship was observed between viral RNA Ct-values and dilutionfactor (where Ct-values increased as the dilution factor increased), atsimilar levels to inoculum only controls, indicating the absence of anymeasurable viral growth after Nasodine treatment and a reduction indetectable virus by at least −3.6 Log10 in the Nasodine samples,representing a reduction in viral RNA copies of over 99.97%.

Conclusion

The objective of this study was to compare and assess the virucidalactivity of PVP-I solution against the newly emerged SARS-CoV-2, thecausative agent of COVID-19 disease. Using real-time TaqMan RT-PCRmethods it was determined that PVP-I possesses clear virucidal activityagainst SARS-CoV-2 and based on the RT-PCR data obtained effectivelyeliminated the replication competent SARS-CoV-2 present in the samplecompared to media alone as evidenced by the lack of viral replication inthe dilutions cultured from PVP-I treated samples compared to theequivalent untreated samples. A comparison of the viral RNA copy numberassociated with cultures derived from the least diluted untreatedcontrol sample with the equivalent PVP-I sample indicated a −3.6 Log10reduction in RNA copies or a >99.97% PVP-I mediated kill. The extent ofthis quantifiable “kill” is limited by the titre of measurable viablevirus in the control sample.

The invention claimed is:
 1. A method of reducing SARS-CoV-2 viral loadin a human subject infected with SARS-CoV-2, the method comprisingapplication to the nasal passages of the human subject 1 to 12 timesdaily commencing after or around the time of exposure to the SARS-CoV-2and/or thereafter for preferably up to 21 days, an effective amount of apharmaceutical preparation comprising povidone-iodine (PVP-I) at aconcentration of greater than 0.10% w/v and less than about 1.25% w/v,wherein the application causes the reduction of the SARS-CoV-2 viralload in the human.
 2. The method as claimed in claim 1 wherein thepharmaceutical preparation is applied to the human subject prior toexposure of the subject to SARS-Co-V-2 or a person infected withSARS-CoV-2.
 3. The method as claimed in claim 2 wherein the humansubject is a healthcare worker who is, or is likely to be, exposed tothe virus or infected patients.
 4. The method as claimed in claim 1,wherein the PVP-I concentration in the pharmaceutical preparationapplied to the nasal passages is about 0.1% to about 1.0% w/v.
 5. Themethod as claimed in claim 1, wherein the PVP-I concentration in thepharmaceutical preparation applied to the nasal passages is about 0.2%to about 0.5% w/v.
 6. The method as claimed in claim 1, wherein thePVP-I concentration in the pharmaceutical preparation applied to thenasal passages is about 0.2% to about 0.45% w/v.
 7. The method asclaimed in claim 1, wherein the pharmaceutical preparation is in adosage form selected from the group consisting of intranasal solutions,liposomal preparations, drops, sprays, gels, aerosols, and inhalants. 8.The method as claimed in claim 1 wherein the pharmaceutical preparationis an aqueous based intranasal preparation where water makes up fromabout 80% w/v to about 96% w/v of the total preparation.
 9. The methodas claimed in claim 1 wherein the pharmaceutical preparationadditionally includes an amount of potassium iodide of from about 0.005to about 0.05% w/v of the total preparation.
 10. The method as claimedin claim 1 wherein the pharmaceutical preparation additionally includesan amount of potassium iodate of from about 0.001 to about 0.03% w/v ofthe total preparation.
 11. The method as claimed in claim 1 wherein thepharmaceutical preparation is an aqueous based intranasal preparationwhere water makes up from about 80% w/v to about 96% w/v of the totalpreparation and has a pH of from about 3-6.
 12. The method as claimed inclaim 1 wherein the pharmaceutical preparation additionally includes anamount of humectant from about 1 to about 10% w/v of the totalpreparation.
 13. The method as claimed in claim 1 wherein thepharmaceutical preparation additionally includes an amount of a polarsolvent from about 0.2 to about 2% w/v of the total preparation.
 14. Themethod as claimed in claim 1 wherein the pharmaceutical preparationadditionally includes an amount of a counter-irritant or localanaesthetic from about 0.001 to about 1% w/v of the total preparation.15. The method as claimed in claim 1 wherein the pharmaceuticalpreparation additionally includes an amount of a preservative from about0.01 to about 0.5% w/v of the total preparation.
 16. The method asclaimed in claim 1 wherein the pharmaceutical preparation additionallyincludes an amount of a buffer from about 0.05 to about 0.5% w/v of thetotal preparation.